The authors acknowledge Don Norwood from Editing Services, Research Medical Library at MD Anderson for editing this article and the Multiplex Immunofluorescence and Image Analysis Laboratory at the Department of Translational Molecular Pathology at MD Anderson. This publication resulted in part from research facilitated by the scientific and financial support for the Cancer Immune Monitoring and Analysis Centers-Cancer Immunologic Data Commons Network (CIMAC-CIDC) provided through the National Cancer Institute (NCI) Cooperative Agreement (U24CA224285) to The University of Texas MD Anderson Cancer Center Cancer Immune Monitoring and Analysis Center (CIMAC).

Tissue samples were obtained for research purposes in accordance with the Institutional Review Board of The University of Texas MD Anderson Cancer Center, and samples were further de-identified.
1. Antibody selection
<ol>
	<li>Define the research questions to choose the best available antibody clones. Use the Human Protein Atlas, published research, and antibody manufacturer websites as research resources. 
	NOTE: The selection of adequate human tissue controls and the ...

<ol>
	<li>Laplane, L., Duluc, D., Bikfalvi, A., Larmonier, N., Pradeu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+tumour+microenvironment.">Beyond the tumour microenvironment.</a> International Journal of Cancer. 145 (10), 2611-2618 (2019).</li><li>Galli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relevance+of+immune+cell+and+tumor+microenvironment+imaging+in+the+new+era+of+immunotherapy.">Relevance of immune cell and tumor microenvironment imaging in the new era of immunotherapy.</a> Journal of Experimental &amp; Clinical Cancer Research. 39 (1), 89 (2020).</li><li>Liu, C. C., Steen, C. B., Newman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+approaches+for+characterizing+the+tumor+immune+microenvironment.">Computational approaches for characterizing the tumor immune microenvironment.</a> Immunology. 158 (2), 70-84 (2019).</li><li>Eisenstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sorting:+Divide+and+conquer.">Cell sorting: Divide and conquer.</a> Nature. 441 (7097), 1179-1185 (2006).</li><li>Scognamiglio, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+immunohistochemistry+assay+to+evaluate+the+melanoma+tumor+microenvironment.">Multiplex immunohistochemistry assay to evaluate the melanoma tumor microenvironment.</a> Methods in Enzymology. 635, 21-31 (2020).</li><li>Guo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+34-marker+panel+for+imaging+mass+cytometric+analysis+of+human+snap-frozen+tissue.">A 34-marker panel for imaging mass cytometric analysis of human snap-frozen tissue.</a> Frontiers in Immunology. 11, 1466 (2020).</li><li>Fu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+architecture+of+the+immune+microenvironment+orchestrates+tumor+immunity+and+therapeutic+response.">Spatial architecture of the immune microenvironment orchestrates tumor immunity and therapeutic response.</a> Journal of Hematology &amp; Oncology. 14 (1), 98 (2021).</li><li>Rost, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+ion+beam+imaging+analysis+for+quantitation+of+protein+expresssion+in+cancer+tissue+sections.">Multiplexed ion beam imaging analysis for quantitation of protein expresssion in cancer tissue sections.</a> Laboratory Investigation. 97 (8), 992-1003 (2017).</li><li>Keren, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structured+tumor-immune+microenvironment+in+triple+negative+breast+cancer+revealed+by+multiplexed+ion+beam+imaging.">A structured tumor-immune microenvironment in triple negative breast cancer revealed by multiplexed ion beam imaging.</a> Cell. 174 (6), 1373-1387 (2018).</li><li>Mathieson, W., Thomas, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+formalin-fixed,+paraffin-embedded+biospecimens+must+be+used+in+genomic+medicine:+An+evidence-based+review+and+conclusion.">Why formalin-fixed, paraffin-embedded biospecimens must be used in genomic medicine: An evidence-based review and conclusion.</a> The Journal of Histochemistry and Cytochemistry. 68 (8), 543-552 (2020).</li><li>Baghban, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironment+complexity+and+therapeutic+implications+at+a+glance.">Tumor microenvironment complexity and therapeutic implications at a glance.</a> Cell Communication and Signaling. 18 (1), 59 (2020).</li><li>Cai, S., Allam, M., Coskun, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+spatial+bioimaging+for+combination+therapy+design.">Multiplex spatial bioimaging for combination therapy design.</a> Trends in Cancer. 6 (10), 813-818 (2020).</li><li>Allam, M., Cai, S., Coskun, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+bioimaging+of+single-cell+spatial+profiles+for+precision+cancer+diagnostics+and+therapeutics.">Multiplex bioimaging of single-cell spatial profiles for precision cancer diagnostics and therapeutics.</a> NPJ Precision Oncology. 4, 11 (2020).</li><li>Rad, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Pandora's+box+of+novel+technologies+that+may+revolutionize+lung+cancer.">The Pandora's box of novel technologies that may revolutionize lung cancer.</a> Lung Cancer. 159, 34-41 (2021).</li><li>Tan, W. C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+multiplex+immunohistochemistry/immunofluorescence+techniques+in+the+era+of+cancer+immunotherapy.">Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy.</a> Cancer Commununications. 40 (4), 135-153 (2020).</li><li>Ptacek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+ion+beam+imaging+(MIBI)+for+characterization+of+the+tumor+microenvironment+across+tumor+types.">Multiplexed ion beam imaging (MIBI) for characterization of the tumor microenvironment across tumor types.</a> Laboratory Investigation. 100 (8), 1111-1123 (2020).</li></ol>

The authors have no conflicts to disclose.

The comprehensive elucidation of the complex, intricate interactions among the multiple components of a TME remains a pivotal objective of cancer research. Manufacturers have introduced numerous multiplexed assays as part of this effort, especially over the past 5 years. Multiplexed spatial analysis is a versatile and powerful tool that facilitates the simultaneous categorization of several targets while preserving structural morphology in tumor samples. Spatial analysis techniques can be performed using fluorescence ima...

The importance of the numerous cell types surrounding clonal tumor populations is a crucial element in the categorization of carcinogenesis. Interest in elucidating this tumor microenvironment (TME) composition and interactions has risen continuously following the establishment of immune-based therapy as part of the cancer treatment arsenal. Therefore, treatment strategies have shifted from a tumor-centric approach to a TME-centric one1.
Efforts to elucidate the roles of immune cells in tumor surveillance and cancer development have increased strikingly in recent years2,3. In medical research, a plethora of methods, including cytometry-based methods and singleplex and multiplex imaging technologies, arose as part of this attempt to decipher the unique interactions of multiple elements of TMEs.
Pioneering methods such as flow cytometry (invented in the 1960s), fluorescence-activated cell sorting, and mass cytometry are focused mainly on identifying and quantifying TME components4. Even though cytometry-based quantitative techniques allow for immune landscape phenotyping, determining the cellular spatial distribution is impossible. Conversely, methods such as standard singleplex immunohistochemistry preserve the tissue architecture and enable researchers to analyze cellular distribution, though a reduced number of targets in a single tissue section is a limitation of these methods5,6. Over the past several years, multiplexed imaging technologies for single-cell resolution such as multiplex immunofluorescence, barcoding fluorescence imaging, and imaging mass spectrometry have emerged as comprehensive strategies for acquiring information on simultaneous marker staining using the same tissue section7.
Here we present a technology that couples metal tagged antibodies and secondary ion mass spectrometry and enables single-cell resolution quantification, marker co-expression (phenotyping), and spatial analysis using formalin-fixed, paraffin-embedded (FFPE), and fresh frozen (FF) tissue samples8,9. FFPE samples are the most widely used materials for tissue archiving samples and represent a more readily available resource for multiplexed imaging technologies than fresh frozen samples10. Additionally, this technology offers the possibility of reacquiring images months after. Herein, we discuss our staining and image processing protocols using FFPE tissue samples.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100% Reagent Alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>R8382</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Reagent Alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>R3404</td>
			<td></td>
		</tr>
		<tr>
			<td>80% Reagent Alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>R3279</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Reagent Alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>R315</td>
			<td></td>
		</tr>
		<tr>
			<td>20X TBS-T</td>
			<td>Ionpath</td>
			<td>567005</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Low-Barium PBS pH 7.4</td>
			<td>Ionpath</td>
			<td>567004</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris pH 8.5&#160;</td>
			<td>Ionpath</td>
			<td>567003</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#176;C Refrigerator</td>
			<td>ThermoScientific</td>
			<td>REVCO</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipette Tips P10</td>
			<td>Olympus</td>
			<td>24-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipette Tips P20</td>
			<td>Olympus</td>
			<td>24-404</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipette Tips P200</td>
			<td>Olympus</td>
			<td>24-412</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Barrier Pipette Tips P1000</td>
			<td>Olympus</td>
			<td>24-430</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal Filter Ultrafree-MC</td>
			<td>Fisher Scientific</td>
			<td>UFC30VV00</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized H2O</td>
			<td>Ionpath</td>
			<td>567002</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyDip Slide Staining Jar, Green</td>
			<td>Electron Microscopy Sciences</td>
			<td>71385-G</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyDip Slide Staining Jar, Yellow</td>
			<td>Electron Microscopy Sciences</td>
			<td>71385-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyDip Slide Staining Kit (Jar+Rack), White</td>
			<td>Electron Microscopy Sciences</td>
			<td>71388-01</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyDip Stainless Steel Holder</td>
			<td>Electron Microscopy Sciences</td>
			<td>71388-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde 70% EM Grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>16360</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Induced Epitope Retrieval (HIER) buffer: 10X Tris with EDTA, pH 9</td>
			<td>Dako</td>
			<td>S2367</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat resistant slide chamber</td>
			<td>Electron Microscopy Sciences</td>
			<td>62705-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic barrier pen</td>
			<td>Fisher</td>
			<td>50-550-221</td>
			<td></td>
		</tr>
		<tr>
			<td>MIBI/O software</td>
			<td>Ionpath</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>MIBIcontrol software</td>
			<td>Ionpath</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>MIBIslide</td>
			<td>Ionpath</td>
			<td>567001</td>
			<td></td>
		</tr>
		<tr>
			<td>MIBIscope</td>
			<td>Ionpath</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5415D</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>RM2135</td>
			<td></td>
		</tr>
		<tr>
			<td>Moisture Chamber (Humid Chamber)</td>
			<td>Simport</td>
			<td>M922-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) Tablets</td>
			<td>Fisher Scientific</td>
			<td>BP2944100</td>
			<td></td>
		</tr>
		<tr>
			<td>PT Module</td>
			<td>Thermo Scientific</td>
			<td>A80400012</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid-Flow Sterile Disposable Filter Units</td>
			<td>Fisher Scientific</td>
			<td>097403A</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>BioRocker</td>
			<td>S2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin column (Ultrafree-MC Spin Filter, 0.5mL 0.1&#956;m )</td>
			<td>MillQ</td>
			<td>UFC30VV00</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide oven</td>
			<td>Fisher Scientific</td>
			<td>6901</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccum Cabinet Desiccator</td>
			<td>VWR</td>
			<td>30621-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Task-whipe</td>
			<td>Kimberly Clark</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>534056-4L</td>
			<td></td>
		</tr>
	</tbody>
</table>

expanding the comprehension of the tumor microenvironment using mass spectrometry imaging of formalin-fixed and paraffin-embedded tissue samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

Tonsil and lung adenocarcinoma TMA tissue sections (5 mm thick) were obtained and placed on the middle of gold-coated slides following the specifications regarding tissue size and secure margins of the slides. Free glass margins of 5 mm and 10 mm between the edge of the tissue and the lateral and inferior borders of the glass slides, respectively, are necessary for optimal staining. The tissue sections were baked overnight in an oven prior to staining to assure proper adherence of the section to the slide. The antibody p...

Watch this Scientific Journal Video about Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples at JoVE.com

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

These protocols enable single cell resolution quantification, marker co-expression and a special analysis using a formalin-fixed and paraffin-embedded and fresh frozen tissue samples to elucidate the roles of immune cells in cancer. This technique is a powerful tool that facilitate the simultaneous characterization of a 40 marker on a single tissue section, widely preserve the structure of the tissue in the samples. The critical step is antibody optimization and validation.Failure to complete the step, can result in low quality images that may compromise a retaining high quality reproducible data and results. Begin by spinning down all the antibody containing tubes at 10, 000 x G for five minutes, add individual antibodies to the blocking buffer and mix well. Do not disturb the bottom of the antibody tube when pipetting it.Filter the antibody panel by pre-wetting a 0.1 micrometer centrifugal filter device with 100 microliters of blocking buffer. Spin the filter at 10, 000 x G for two minutes and remove the blocking buffer flow-through with a pipette. Then transfer the antibody panel into a spin column and spin the filter at 10, 000 x G for two minutes.Discard the spin column and use the flow-through as the filtered antibody panel. For the antibody staining, remove the blocking buffer from the slides by tipping each slide on its side and gently tapping the edges against a task wiper. Place the slides in a moisture chamber and add 100 to 200 microliters of antibody master mix to the tissue.Add positive and negative control slides to the staining batch and incubate the slides at 4 degrees Celsius overnight. To set up the instrument, login to the control software, and click on Wake Up, if the instrument is in sleep mode. Click on Exchange Sample to load a slide and then click on Continue to open the door.Put the slide in the loading slot with a sample facing up and the label on the right, click on Continue to close the door. Select the project and then select the slide name for the loaded sample. Visualize the panoramic image on the slide optical image pane for the field of view or FOV selection, click on the Mode menu and select Secondary Electron Detector, then click on the Imaging Mode menu and pick QC 300 micrometer.Click on Jog Stage to control the FOV location and then click on the arrows to navigate and select the position of the FOV. Click add FOV, set 400 micrometer by 400 micrometer as the field size and select Fine as the Imaging Mode and 1 millisecond of Dwell Time, click on Confirm. After creating an FOV list, proceed to the image focus and adjust the Stigmation by moving the beam to a tissue region that will not be imaged.Adjust the parameters until a clear central image is obtained, click on Start Run. The acquired images will be automatically uploaded and stored in the web-based image management application. The matrix shown here describes the percent crosstalk derived from the probe purity and oxides.Here blue boxes indicate greater than or equal to 0.5%crosstalk and clear boxes indicate less than 0.5%crosstalk. For the Mass tags, probes that contributed at least 0.5%crosstalk into no channels, one or two channels, or more than two channels are shown here. Mass channels receiving at least 0.5%crosstalk from no probes, one or two probes, or more than two probes are also shown.The representative staining images in different tissues like lung adenocarcinoma, non-neoplastic kidney and tonsil are depicted here. An image of a tonsil tissue section stained using this technique and visualized using a third party digital image analysis software program before filtering and correction is presented here. The image of the same section under the same conditions after filtering and correction steps is shown here.This method generate images that can be uploaded to a third party digital image analysis software, allowing comprehensive analysis such as tissue compartmentalization, cell segmentation, marker co-expression, as well as neighbor analysis and distant analysis. Highly multiplex imaging methods are available through research. By constructing different panels, researchers can investigate cancer, normal, paraneoplastic, inflammatory and infectious samples.

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Research

JoVE Journal

Cancer Research

2.0K visualizações.  The University of Texas MD Anderson Cancer Center. Esses protocolos permitem quantificação de resolução de células únicas, co-expressão de marcadores e uma análise especial usando amostras de tecido congelado com estatação fixa e parafina parafina para elucidar os papéis das células imunes no câncer. Esta técnica é uma ferramenta poderosa que facilita a caracterização simultânea de um marcador 40 em uma única seção de tecido, preservando amplamente a estrutura do tecido nas amostras. O passo crítico é a otimização e v...

Vídeo: Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples